viral modulation of tlrs and cytokines and the related … · 2019. 7. 30. · review article viral...

Review ArticleViral Modulation of TLRs and Cytokines and the RelatedImmunotherapies for HPV-Associated Cancers

Marconi Rego Barros Jr ,1 Talita Helena Araújo de Oliveira,1

Cristiane Moutinho Lagos de Melo ,2 Aldo Venuti ,3,4 and Antonio Carlos de Freitas 1

1Laboratory of Molecular Studies and Experimental Therapy (LEMTE), Department of Genetics, Center of Biological Sciences,Federal University of Pernambuco, Av. Prof Moraes Rego, 1235 Cidade Universitária 50670-901 Recife, PE, Brazil2Laboratory of Immunological and Antitumor Analysis (LAIA), Department of Antibiotics, Center of Biological Sciences, FederalUniversity of Pernambuco, Av. Prof Artur de Sá, s/n, Cidade Universitária 50740-525 Recife, PE, Brazil3Tumor Immunology and Immunotherapy Unit, Department of Research, Advanced Diagnostic and Technological Innovation,Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144 Roma, Italy4HPV-Unit, UOSD Tumor Immunology and Immunotherapy, Regina Elena National Cancer Institute, Via Elio Chianesi 53,00144 Roma, Italy

Correspondence should be addressed to Aldo Venuti; [email protected] Antonio Carlos de Freitas; [email protected]

Received 18 December 2017; Accepted 26 March 2018; Published 2 May 2018

Academic Editor: Andréia M. Cardoso

Copyright © 2018 Marconi Rego Barros Jr et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalwork is properly cited.

The modulation of the host innate immune system is a well-established carcinogenesis feature of several tumors, includinghuman papillomavirus- (HPV-) related cancers. This virus is able to interrupt the initial events of the immune response,including the expression of Toll-like receptors (TLRs), cytokines, and inflammation. Both TLRs and cytokines play acentral role in HPV recognition, cell maturation and differentiation as well as immune signalling. Therefore, the imbalanceof this sensitive control of the immune response is a key factor for developing immunotherapies, which strengthen thehost immune system to accomplish an efficient defence against HPV and HPV-infected cells. Based on this, the review isaimed at exposing the HPV immune evasion mechanisms involving TLRs and cytokines and at discussing existing andpotential immunotherapeutic TLR- and cytokine-related tools.

1. Introduction

The evasion of immune tumor surveillance is a well-established feature of cancer [1]. In HPV-related tumors,papillomavirus is responsible for this escape (Figure 1). Thisvirus is able to abrogate the initial steps of the immune innatesystem, which embraces Toll-like receptor signalling as wellas cytokine synthesis and secretion, thus, compromising theimmune response against an invasive agent [2].

TLRs and cytokines play pivotal roles in the immunedefence against HPV-infected and tumor cells. TLRs, forexample, are responsible for recognizing the conservedpathogen-associatedmolecularpatterns (PAMPs), promoting

changes onhost endogenous ligands and thus, initiating a pro-tein cascade that follows in the expression of keymolecules forthe development of the immune response, which includes thesynthesis and secretion of cytokines [3]. Cytokines, in turn,are important mediators of immune cell activities, suchas cell recruitment, maturation, and signalling [4]. In addi-tion, both molecules (TLRs and cytokines) control geneexpression and are essential for creating a suitable tumormicroenvironment, either for immune surveillance or forimmune modulation. As a result, these molecules areinvolved in the pathogenesis of various diseases besidescancer, such as autoimmune, inflammatory, and infectiousdiseases [5].

HindawiJournal of Immunology ResearchVolume 2018, Article ID 2912671, 17 pageshttps://doi.org/10.1155/2018/2912671

http://orcid.org/0000-0003-1180-5712

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http://orcid.org/0000-0003-2322-6857

http://orcid.org/0000-0002-4957-9549

https://doi.org/10.1155/2018/2912671

Therefore, both TLRs and cytokines are crucial targets forimmunotherapeutic studies that are aimed at preventing ortreating cancer. In fact, they have already been used inpharmaceutical formulations for cancer therapies by takingadvantage of the fact that currently there is no effectivetreatment for HPV-related cancer patients, especially for thosewho have unsuccessfully undergone radio- and chemotherapytreatments. Several major studies have reported the greatpotential value of immunotherapeutic approaches that modu-lated TLR and cytokine levels or synthesis [6, 7]. In accordancewith these studies, this review highlights the immune mecha-nisms of TLRs and cytokines for infection resolution and viralimmune evasion activities correlated with HPV-associatedcancers. Furthermore, we will discuss the effectiveness of theimmunotherapeutic approaches involving these targets.

2. Toll-Like Receptors

TLRs are specialized receptors which detect PAMPs anddamage- or danger-associated molecular patterns (DAMPs).They are found in immune (e.g., APCs, natural killer) andnonimmune (e.g., stromal, epithelial, and cancer) cells onthe plasma membrane (e.g., TLRs 1, 2, 4, 5, 6, and 10) andon the surface of organelles, such as endosomes, lysosomes,and endoplasmic reticulum (e.g., TLRs 3, 7, 8, and 9) [5].

The molecular structure of these receptors is comprisedof two domains: the extracellular N-terminal and theintracellular C-terminal domains. The extracellular domaincontains leucine-rich repeats responsible for recognition ofPAMPs, depending on the TLR subtype; the C-terminal por-tion has a conserved region called Toll/IL-1 receptor (TIR)domain [5], which is responsible for transducing the signalfor adapter molecules.

Usually, TLRs are associated with the protection againstpathogen invasion, carcinogenesis, and infection clearance,which are essential in inducing and linking innate and adap-tive responses, such as the Th1 and the cytotoxic cell-mediated subtypes [3]. In addition, TLRs are able to recognizesome host endogenous ligands (see Figure 2), representing animportant role in tissue repair and homeostasis [9].

TLRs support the uptake, processing, and presentation ofantigens by APCs, boost DCmaturation, NK cell cytotoxicity,and targeted cell apoptosis aswell as upregulate the expressionofmajor histocompatibility complex (MHC), C-C chemokinereceptor 7 (CCR7), interferons (e.g., IFN-I, IFN-γ), andinflammatory cytokines (e.g., IL-6, IL-12) [3]. Nevertheless,several TLRs were reported to be overexpressed in cancer.They were associated with malignant transformation by pre-venting the activation of immune responses or enhancinginflammation through the induction of the nuclear transcrip-tion factor kappa B (NF-κB) pathway. Therefore, TLRs seemto have dual functions in the tumor microenvironment, tothe extent that even cancer cells may express those moleculesin order to alter immune response and sustainmalignant pro-gression [3, 10]. Indeed, recently, TLRs were showed to acti-vate the nitric oxide signalling pathway, supporting cervicalcarcinogenesis [11].

The expression of both TLR4 and 9 receptors wasreported to be altered during HPV infection. In cervical carci-nogenesis studies, TLR4 and 9 levels were reported to be cru-cial for the initiation of the innate immune response due tothe induction of cytokine synthesis and cytotoxicity on targetcells. As a consequence, lower levels are generally associatedwith a poor prognosis and cancer progression [8, 12, 13].However, these receptors have also been correlated withmalignancy [3, 11, 14, 15]. TLR4 was found to be overex-pressed in human cervical cancer line (HeLa) [11, 14] as well

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Figure 1: Th1 immune evasion induced by HPV in tumor microenvironment. HPV (1) interferes in TLR expression and in immunesignalling pathways; (2) suppresses IL-12 expression, leading to a decreased production of IFNs (type I and IFN-γ) and blockingmacrophages (M1 phenotype) and dendritic cell (DC) activity; (3) promotes downregulation of HLA expression and transportation tomembrane surface, preventing antigen presentation, T cell activation, and NK and CTL cytotoxicity; and (4) finally, the downregulation oflymphocyte activity related to a decreased activity of APC cells (as M1 and DC) impairs adaptive immune activation [2].

2 Journal of Immunology Research

as in premalignant and malignant specimens [16]. It was alsoassociated with the proliferation of cancer cells and immuno-suppression, through the production of IL-6, TGF-β, andother immunomodulatory cytokines [3, 10]. TLR9 was alsoshown to be overexpressed in high-grade cervical lesionsand/or cancer [3, 13, 15–17]. It is possible that the upregula-tion of these receptors in malignant lesions may be due tothe following components: (i) compensation for TLR defi-ciency or by harnessing mechanisms of the host immunedefence system against tumor cells, (ii) reflecting increasedinflammation, which damages an effective immune responseagainst the pathogen, (iii) the existence of polymorphismsand the measurement of different subtypes, which misleadsdata interpretation, (iv) the measurements at different inter-vals during cancer progression, (v) the differences in methodsof TLR assessment in cell lines, or (vi) the activity of tumorcells that create a proper milieu for cancer development.

Modulation of TLR9 levels probably occurs due toHPV16 E7 oncoprotein activity on the TLR9 promoter byinterference in the NF-κB pathway. A chromatin repressivecomplex was also found on the TLR9 promoter, negativelyregulating its transcription (Figure 3) [8]. A repression ofthe TLR9 expression disturbs the synthesis of IL-6, IL-8,and C-C chemokine ligand 20/macrophage inflammatoryprotein-3α (CCL20/MIP-3α) (which is an important chemo-kine for immune surveillance because of the recruitment oflymphocytes and Langerhans cells (LCs) to skin) [18]. Theviral transcription and replication processes also undergointervention, due to interferon deficiency caused by TLR9repression [8]. Moreover, the altered levels of TLR9 can becaused by the devious expression of specific polymorphismsthat change the effective receptor availability [19].

Other TLR expressions were also notably altered. In SCC(squamous cell carcinoma) specimens, it was demonstratedthat the TLRs 3, 4, and 5 were significantly underexpressed

while TLR1 was the only significantly overexpressed (TLRs2, 7, 8, and 9 were not significant) when compared to the nor-mal samples [13]. However, opposite results were alsoobserved [3, 10, 11, 15, 16, 20]. No study has clearly demon-strated that altered TLR levels are a response of the hostimmune systemagainst the infection or a consequence of virusactivity supporting the infection. In addition, the knowledgeof which cell is responsible for the alteration of TLR levelswould be crucial to understand the TLRs’ role in carcinogene-sis; immune, stromal, and cancer cells have different functionsin cancer development and thus, the shift of their TLR expres-sion pattern could represent a marker for cancer progressionor resolution [15].

Besides, another study found similar results and showedthat the mRNA expression of TLR7 and 8 in cervical biopsiesof cervical cancer patients was elevated [2]. In turn, infectionregression (HPV16) was also associated with an increasedexpression of several TLRs (3, 7, 8, and 9), and their modula-tion could be used as a therapeutic approach [18].

Regarding other HPV-related cancers, TLRs were not asextensively studied like in cervical cancer. HPV is a key etio-logical factor of head and neck squamous cell carcinoma(HNSCC), in particular of oropharyngeal squamous cellcarcinoma (OPSCC). HNSCC is commonly recognized asan immunosuppressive disease due to HPV activity. Conse-quently, an imbalanced cytokine profile, low amounts ofCD3+ CD4+ and CD8+ T lymphocytes, high infiltration ofM2 macrophages (TAM), Treg cells and Treg/CD8+ T cellratio, impaired NK cell activity (higher expression levels ofKIR genes), augmented expression of inhibitory receptors(e.g., CTLA-4, LAG-3, TIM-3, and PD-1), and decreasedantigen presentation have been observed [21, 22].

Thereafter, the expression levels of several TLRs werereported and its dubious role was evident in some cases. Itwas found that TLR2 was upregulated in both vicinity

Microbial ligands

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Figure 2: TLR activation. The scheme shows major microbial and endogenous ligands that activate TLR signals in immune cell surface. Thesesignals are able to promote host protection against pathogen invasion and infection establishment [5].

3Journal of Immunology Research

immune cells and malignant keratinocytes in the microenvi-ronment of oral squamous cell carcinoma (OSCC) comparedto hyperplasia [23]. In another OSCC study, TLR3 was foundto be upregulated in three head and neck cell lines (mRNAand protein), as well as when it induced apoptosis of thetumor cells, and in vivo, when it interrupted tumor growth.Also in this study, the mRNA expression of other TLRs wasassessed observing elevated expressions of TLRs 1, 2, 4, and6 while the TLRs 5, 7, 8, and 10 were found reduced [24].In this type of carcinoma, however, TLR3 was found to haveelevated levels and this event was attributed to an increase oftumor aggressiveness and invasion [25], a similar conclusionof another study which measured TLR3 in various HNSCCcell lines [26]. Both studies associated the triggering of theNF-κB pathway with the increase in tumor aggressiveness.In the mentioned studies above, it was not verified whetherHPV was present or not.

TLR4 and 9 were also assessed in OSCC, and these recep-tors were found to have elevated expression levels which werecorrelated with tumor development [10]. Whether theincreased levels of these receptors were associated with carci-nogenesis or a reaction of the host immune response againsttumor cells is still not known. TLR4 was also correlated withtumor development and protection of cancer cells from host

immune response in an HNSCC study [27]. Several otherstudies have also reported controversial results regardingOSCC and HNSCC [10, 28], though it has been observed thatdifferent cell lines were used several times for the same pur-pose, which could explain the opposite results in some cases.

TLR7 was found overexpressed in the nuclear membraneand nuclei of cancer cells (a novel localization discovered)having higher levels in HPV-positive specimens, unlikeTLR9 that showed reduced expression levels in HPV-positive samples compared to HPV-negative. TLR7 alsoshowed altered localization depending on the cancer cell sta-tus: it was found with elevated levels in the plasma membraneof cancer-free cells compared to cancer cells, where thisreceptor was observed to have higher levels in the nuclearmembrane and nuclei. Therefore, it seems that TLR7 mayplay different roles depending on the cancer cell status. Fur-thermore, increased levels in TLR7 demonstrated to be statis-tically significant for the direct correlation with p16. Thus,the increased levels of TLR7 could be indirectly related toE7 expression since the elevated levels of p16 are caused bythe deregulation of pRb pathway, initially caused by E7 onco-genic activity, which probably also altered TLR9 levels. Theother TLRs did not show any significant differences betweencancer and control groups [29].

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Figure 3: Inhibition of TLR9 expression by HPV16 E7 oncogene takes place via NF-κB canonical pathway, when this oncoprotein recruits theinhibitory complex NF-κB p50–p65 to a new cis element at the TLR9 promoter. This occurs with the additional binding of ERα (estrogen α) toanother neighbour cis element, ERE (estrogen-responsive element), within that same promoter, and in the presence of HPV16 E7. ERα is alsoable to interact with the p65 subunit in the peri- or intranuclear region and contribute to transcription repression. Furthermore, it was alsoobserved that there was a chromatin repressive complex composed by JARID1B demethylase and by HDAC1 deacetylase. These two catalyticunits interact with ERα and negatively regulate TLR9 expression. The consequence of preventing TLR9 expression is the establishment of animmunosuppressive status with the inhibition of interferon and immune surveillance by cytokine responses [8]. NF-κB blue circlecorresponds to p50 subunit and the purple one to p65. The straight blue arrows indicate an activation process or progress to the nextstage; the curved arrows indicate motion; and the progressive arrows indicate the movement of some molecules interacting with the target.IKK: inhibitor of kappa B kinase; P: phosphate group; M: methyl group; A: adenyl group; JARID1B: lysine-specific demethylase 5B;HDAC1: histone deacetylase 1; Site B: 9-10 base pair DNA sites where p50 and p65 subunits bind.


Based on what has been discussed above, the modulationof TLR expression or activities has been considered in thetreatment of cervical and head and neck carcinomas as adju-vants in various vaccine strategies. The goal is to increase thesynthesis of cytokines (e.g., IFN, TNF-α) and chemokines,generally by activatingNK and dendritic cells, so as to activateCTL cells for generation of effector responses [30]. For amoreefficient outcome, TLR agonists can be used in combinationwith non-TLR agonists, as demonstrated in a C57BL/6 micetumor model in which HPV16 E7 vaccine was coadmini-strated with monophosphoryl lipid A (MPL), a TLR4 agonist,and α-galactosylceramide [7]. Also, TLR activities can beblocked aiming to hamper inflammation by preventing TLRdownstream activation signalling, such as the MyD88-NF-κB pathway. Another interesting view to be highlighted isthe importance of the modulation of TLR expression in stro-mal cells. It was found that these receptors were upregulatedin these cells and may contribute to carcinogenesis [15].

Accordingly, several pharmacologic substances, whichmodify the activity of these receptors, have been tested/usedfirst as adjuvants in vaccines for cervical cancer (e.g., Cervarix)and later in other HPV-related cancers. In the Cervarix vac-cine for example, MPL is used to activate the innate responseby interferon and proinflammatory cytokine synthesis. As aconsequence, the adaptive response is also induced. Otherexamples are CpG (TLR9 agonist) alone or with rlipo (TLR2agonist) [31], imiquimod and resiquimod (both TLR7 ago-nists), poly(I:C) (TLR3 agonist), and VTX-2337 (TLR8 ago-nist). Many studies have shown satisfactory results,especially with the simultaneous use of these agonists. Gener-ally, an increased rate of tumor cell death upon TLR stimula-tion was observed [30]. Also, very high percentages of curingpreexisting tumors in mice were reported [31–33].

InHPV-related cancers, the use of these adjuvants has alsobrought better outcomes. Imiquimod is able to induce Th1responses by stimulating DC maturation and migration,Langerhans cell migration to draining lymph nodes, the inhi-bition of myeloid-derived suppressor cells, and the secretionof interleukins and cytokines [34]. In another study, imiqui-mod and poly(I:C) effects on cell death in in vitro and in vivoHNSCC models were evaluated. Both agonists were found tocause an increase in tumor cell death in vitro and in vivo. Inaddition, poly(I:C) induced higher levels of proinflammatorycytokine secretion (IFN-I, IL-6, and CXCL-10), MHC Iexpression of tumor cells, and monocyte activation in vitro,impairing tumor growth in vitro and in vivo even when TLRsignalling was hampered on host cells [35]. Another studyis currently recruiting participants to evaluate polyICLC(a modified version of poly(I:C)) combined with tremeli-mumab, a CTLA-4 antibody, and durvalumab, a PD-L1 anti-body (clinicaltrials.gov identifier NCT02643303).

Another example is motolimod (VTX-2337), a TLR8agonist which was tested for the treatment of HNSCC andother tumors. It was seen that this substance caused anincrease in antitumor activities by inducing cytokine andchemokine synthesis as well as activating monocytes, NK,and dendritic cells, consequently boosting T cell activation[36, 37]. In a study using the TLR8 agonist with an anti-EGFR monoclonal antibody (cetuximab), an increase in NK

cell-mediated cancer cell lysis and an enhancement of DCcross-priming of EGFR-specific CD8+ T cells were observed[38]. Other possibilities are currently being tested for VTX-2337, such as combining it with cisplatin or carboplatin/fluo-rouracil/cetuximab (NCT01836029) and with cetuximab orcetuximab and nivolumab, an anti-PD-1 antibody(NCT02124850). In regard to TLR4, OK-432 (picibanil,approved in Japan) was tested in association with DCs andchemotherapy, and the results still have not been reported(NCT01149902) [30].

Other studies have also been or are currently beingconducted with other TLRs: ISA201 (HESPECTA, a secondgeneration vaccine based on ISA101), which uses a syntheticTLR2 agonist (Amplivant) with two HPV16 E6 peptides(NCT02821494) [38]; EMD 1201081 (IMO2055, TLR9agonist) + cetuximab (NCT01040832), which did not demon-strate additional clinical efficacy than using cetuximab alonebut itwaswell tolerated bypatients [39]; EMD1201081+fluo-rouracil + cisplatin + cetuximab (NCT01360827); and entoli-mod (CBLB502), a TLR5 agonist which is being tested incombination with cisplatin and radiation (NCT01728480)due to the previously shown effects on radiation: it inducedan increase in its therapeutic effect and a reduction of its toxic-ity in vivo when administered 1 h after exposure to radiation[40]. Table 1 summarizes the mentioned studies and is corre-lated with the Figure 4 which shows the therapeutic role andthe activated signalling pathways of natural and syntheticTLR ligands.

3. Cytokines

Cytokines are the primordial mediators of the immuneresponse, including antitumor activities. It has been reportedthat in cervical cancer as well as in HNSCC, immunostimula-tory signals (Th1 cytokine profile) are hampered whereasproinflammatory and immunosuppressor ones (Th2cytokine profile) are stimulated. Several studies havereported this shift of the cytokine pattern in preneoplasticand cancer specimens [41–43].

It is known that Th1 cytokines are potent activators ofcellular-mediated immunity response that may precedeHPV clearance, while Th2 cytokines impair the immuneresponse, leading to an inefficient virus elimination and tochronic infection [43]. Furthermore, different hrHPV geno-types are associated with different cytokine profiles, so theyinterfere distinctly with the immune system, making diseaseprogression different among the various hrHPV subtypeinfections [44]. Therefore, the modulation of cytokineexpression is a key event for the induction of chronic infec-tion and cancer development. It is known that the appropri-ate cytokine pattern defines the appropriate phenotype ofimmune cells, which whether or not results in the eliminationof infected and (pre)cancerous cells.

Thus, cytokines were widely used in cancer immunother-apy, including cervical cancer and HNSCC. The main goal oftheir use was to induce a CTL response supporting the cancercell apoptosis and tumor regression. They can be used incombination with several immunotherapy approaches suchas DNA, DC-based and protein-based vaccines, TLR


https://clinicaltrials.gov/ct2/show/NCT02643303








agonists, and monoclonal antibodies (e.g., cetuximab and theimmune checkpoint inhibitors like tremelimumab and dur-valumab) [6, 34]. In the next subheadings, the most impor-tant cytokines for the treatment of HPV-related cancers arediscussed according to their roles in the immune response.

3.1. Immunostimulatory Cytokines. Among the immunosti-mulatory cytokines, IL-2, IL-12, TNF-α, and interferonsare the most prominent in anti-infection and antitumoractivity. IL-12 is secreted by activated DCs and macrophagesand is the most effective and promising cytokine for cervicalcancer treatment. Several antitumor activities in animalmodels have been observed [34], such as the increase inIFN-γ and TNF-α levels, maturation of APCs and the lysisof immature ones, and the activation of NK responses(caused by the upregulation of NK activation receptors andligands). Consequently, IL-12 stimulates Th1 polarizationand CTL (antigen-specific response) cytotoxicity and playsan important antiangiogenic role [45].

As a result, IL-12 has been suggested to be used in severalHPV-related cancer treatment strategies for potentiation ofantitumor activity, for instance, in viral gene therapy, coad-ministrated with other cytokines or in DNA vaccine prepara-tions [34, 38, 45], such as the INO-3112 vaccine. Thispromising strategy, which combines the gene sequences ofE6+E7 antigens (VGX-3100) and the IL-12 (INO-9012),has been tested for treatment of both cervical invasive(NCT02172911) and head and neck (NCT02163057) HPV-related cancers in clinical phase I/II trials with good resultsabout safety and CD8+ T cell immunogenicity [46].

Another ongoing study evaluated the immunotherapeu-tic effects of the coadministration of the recombinant IL-12with cetuximab (NCT01468896) in patients with relapse,metastatic, or inoperable HNSCC. In the previous study

regarding this combined treatment, an improvement in thelysis of tumor cell by NK cells was observed. In anotherapproach using IL-12, a higher lymphocyte infiltrate and animproved overall survival rate were observed when IL-12was coadministrated with IL-1β, IFN-γ, and TNF-α [47].

TNF-α is another key cytokine which creates an antitu-moral milieu for virus elimination. This molecule supportsthe activation of macrophages, dendritic and NK cells andrecruits them to the tumor site by inducing keratinocytes torelease MIP-3α [18] and CCL2/MCP-1 (C-C motif chemo-kine ligand 2/monocyte chemoattractant protein-1), whichis reduced in high-grade cervical lesions and in E6/E7-expressing cells [48]. Moreover, it inhibits HPV E6 and E7oncogene transcription and proliferation in HPV-transformed keratinocytes in vitro [49] and causes apoptosisof cervical cancer cell lineage [50]. This cytokine is associatedwith lesion regression; its reduced level in serum and its pres-ence in cervical cancer are correlated with tumor growth [51,52]. Increased levels of TNF-α were also associated withCIN2/3 lesions, but also with an exacerbated inflammatoryresponse [18]. A precise level of inflammatory response,which includes the precise secretion level of several cytokinesincluding TNF-α, is the threshold between the occurrenceand regression of cellular transformation. A sufficient levelof this response at the infection onset is a mark of a validimmune response, but when the inflammatory responsebecomes excessive and persistent, it favours an appropriatemilieu for infection development.

Along with TNF-α, interferon belongs to the group ofcrucial cytokines which creates an antiviral state, activatingcell-mediated immunity which is potentiated in the presenceof TNF-α (see Table 2 for IFN activities). Interferon is classi-fied in type I (IFN-α and -β), type II (IFN-γ), and type III(IFN-λ) [62]. Similar to TNF-α, the amount of interferon

Table 1: TLR-associated immunotherapy approaches for HNSCC treatment.

Number inFigure 4

Therapy Therapy approach StageClinical trialidentifier

Reference

1, 2Imiquimod+ poly(I:C)

TLR7 agonist + TLR3 agonistIn vitro/in vivo

— Klein et al. [35]

2 Poly(I:C) TLR3 agonist + tremelimumab + durvalumab Phase I/II NCT02643303No study wasreported yet.

3 VTX-2337

TLR8 agonist Phase I NCT00688415 Dietsch et al. [37]

TLR8 agonist + cetuximab Phase I/II NCT01334177Stephenson et al.

(2013)and Chow et al. [36]

TLR8 agonist + cisplatin or +carboplatin/fluorouracil/cetuximab

Phase II NCT01836029No study wasreported yet.

4OK-432(Picibanil)

TLR4 agonist Phase I NCT01149902 Galluzzi et al. [30]

5 ISA201 TLR2 agonist + 2 HPV16 E6 peptides Phase I NCT02821494 Bann et al. [38]

6 EMD 1201081TLR9 agonist which was tested with cetuximab Phase II NCT01040832 Ruzsa et al. [39]

TLR9 agonist + fluorouracil + cisplatin+ cetuximab

Phase I NCT01360827No study wasreported yet.

7CBLB502(entolimod)

TLR5 agonist Phase I NCT01728480 Toshkov et al. [40]














arises mainly from NK cells [63], but T cells also synthesizethese cytokines, both cytokines are upregulated in therapeu-tic approaches which cause infection regression [34].

With regard to this, it was suggested that the upregula-tion of IFN would be a good hallmark for HPV16 infectionclearance [64]. This event is generally associated with theexpansion of NK cell cytotoxicity [65] and the expression ofIL-12 and TNF-α in infiltrated proinflammatory lympho-cytes [57]. The increased levels of IFN restore immune func-tion [55] and induce CD4+ and CD8+ responses, which led toa complete regression of the disease in a half of the patientswho had undergone treatment. Several reports have shownthe inhibition of type I IFN expression or their transduction

pathways by HPV16 and 18 oncoproteins (E6 and E7). In thisway, key genes involved in the immune surveillance andcytotoxic response are blocked [2, 56].

Diminished IFN-γ synthesis has also been associatedwith persistent infection and the level of malignancy of cervi-cal lesions [66] as well as to the development of HPV-relatedcancer. In HNSCC, for example, the reduced levels of thiscytokine are able to modify STAT1/STAT3 balance thatblocks antigen presentation and DC maturation [47].

IFN-γ became a marker of the Th1-type antigen-specificcellular immune responses, involved in tumor resolution [67]and HPV clearance [18]. This cytokine induces: (i) synthesisof antiangiogenic factors (e.g., IP-10); (ii) cell cytotoxicity by

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NF-�휅B

NF-�휅B

Figure 4: TLR signalling transducing pathways and the related immunotherapy approaches. Toll-like receptors are expressed in bothimmune and tumor cells, making difficult to decipher their function in cancer. Thus, their modulation in tumor microenvironment resultsfrom a complex balance of host and infected/tumor cell mechanisms which affects their expression and activation. They are activated byspecific ligands (for each TLR) and synthetic substances as showed in the figure: (1) single-stranded RNA and imiquimod for TLR7, (2)double-stranded RNA and poly(I:C) for TLR3, (3) ssRNA and VTX-2337 for TLR8, (4) LPS, lipoteichoic acid, and Picibanil for TLR4, (5)∗lipoproteins, peptidoglycans, lipoteichoic acids, zymosan, mannan, tGPI-mucin, and ISA201 for TLR2, (6) unmethylated dinucleotidecytosine-guanine and EMD 1201081 for TLR9, and (7) flagellin and CBLB502 for TLR5. Once activated, the signal transduction dependson adapter molecules such as MyD88, TIRAP, TRIF, and TRAM in order to activate the transcription of type I interferons and TLR-induced genes. All TLRs, except TLR3, require MyD88 for propagation of their signals. TLR4 requires myeloid differentiation factor-2(MD-2) as a coactivator for its activation by LPS binding [3, 5]. TIRAP: TIR-domain-containing adaptor protein; MyD88: myeloiddifferentiation primary response protein 88; IRAK: IL-1R-associated kinase; TRAM: Toll receptor-associated molecule; TRIF: TIR-domain-containing adapter-inducing interferon-β; TRAF: TNF receptor-associated factor; RIP-1: receptor-interacting protein kinase-1;TAK: TNF receptor-associated factor; TAB: TGF-β-activated kinase I/MAP3K7-binding protein; IKK: inhibitor of kappa B kinase; IκBα:nuclear factor of kappa B, alpha; IRF: interferon regulatory factor; U: ubiquitination; P: phosphorylation.


boosting the overexpression of adhesion molecules; (iii)antigen presentation and synthesis of IL-12 by activationof DC; and (iv) more sensitivity to granule activity anddeath signalling [68]. In addition, type III IFN (IFN-λ)has also been demonstrated to play an important role inimmune response against HPV infection. Alteration of thisinterferon was reported in hrHPV cell infection status, andit was suggested that it may support immune surveillance

against HPV infection and cervical carcinogenesis. IFN-λshows similar activities of type I IFNs but only specificcells respond to this type of IFNs like epithelial cells[62]. For all these activities and owing to the oncoproteininterference with these molecules, interferons are a class ofcytokines with a great potential value when used in thedevelopment of more efficient approaches for HPV-related cancer therapy.

Table 2: IFN immune activities and HPV interferences.

IFN-I (IFN-α/β)

Activities

IFN-α inhibits keratinocyte immortalization induced by HPV16 [53]

Boosts IFN-γ secretion [54] and Th1 response [55]

Induces antibody production [54]

Induces resistance to viral replication [56]

Induces MHC class I and II expression [54]

Induces the activation of NK cells [56]

Plays antiangiogenic and antiproliferative activities [57]

Induces DC maturation and T cell proliferation and priming [54]

Turns virus-infected cells more susceptible for CTL killing [56]

Alters the B cell isotype and differentiation into plasma cell [54]

Prevents T cell apoptosis [54]

Promotes the proliferation of memory T cells [54]

HPV interferences

Its signal transduction pathways are prevented by E6 and E7 activities [18]

IFN-α signalling is inhibited by (i) HPV18 E6 interaction with Tyk2 (tyrosine kinase 2),(ii) E6 binding to IRF-3 which prevents IFN-α transcription, and

(iii) HPV16 E7 prevention of the displacement of p48(subunit of interferon-stimulated gene factor 3 (ISGF3)) to the nucleus

HPV16 E7 also inhibits IRF-1-mediated IFN-β transcription by physically interacting with IRF-1

IFN-α and IFN-β are also downregulated by E6-mediated inhibition of STAT1binding to ISRE and prevention STAT1 expression [57]

IFN-II (IFN-γ)

Activities

IFN-γ as well as IFN-I inhibit transcription of E6/E7 genes in immortalizedkeratinocytes and malignant cells [48]

Upregulates MHC class I in immune and tumor cells [58] as well asMHC I/II in epithelial cells [18]

Promotes differentiation to the Th1 profile and induces these cells to produce IFN-γ [59]

Plays antiviral, antiproliferative [58], and tumor cell killing activities, being associated withlesion regression [60]

Boosts the synthesis of inducible nitric oxide synthase, IP-10 (protein 10 inducible by IFN-γ) and Mig(monocin inducible by IFN-γ) chemokines by macrophages, which augment cytotoxic response [58]

Induces NK cell infiltration and activation [58]

Induces TAP-1 (transporter antigen processing-1) and MCP-1 expression, which are important forT cell antigen recognition and chemoattraction, respectively [58]

HPV interferences

The expression of TAP-1 and MCP-1 is prevented by E7 and E6/E7, respectively [48]

IFN-III (IFN-λ)

Activities

Prevents several tumor cell lines growth [61]

Promotes antiviral and antitumor responses [61]

In the treatment of viral and neoplastic diseases it has been tested with type I IFNs showingsynergic effects and reduced side effects [61]

HPV interferences

None reported


Another cytokinedemonstrating tohave strongantitumoractivity is IL-2,which is producedprimarily byCD4+ cells afterantigenpriming. It plays several important roles, including theactivation andmaturation of DC, stimulation of NK cell cyto-toxicity, the expansion of CD8+ andCD4+ cells, and the polar-ization for the Th1 cell profile [69]. Several studies haveattributed the reduced levels of this cytokine to the lesion pro-gression or cancer in HPV infection [21, 42, 70, 71]. Itsreduced levels were suggested to be a viral evasionmechanism[71], which is frequently associatedwith increased levels of IL-10, TGF-β, and Treg cells [43]. Due to its strong immunopro-tective activity, IL-2 has been used in several immunothera-peutic approaches, like in association with TG4001 HPVvaccine, which is constituted of a recombinant Ankara vac-cinia virus expressing HPV16 E6 and E7 [6]. The disadvan-tages of IL-2 administration are systemic toxicity and theinduction of Treg cell proliferation [69]. For example, inHNSCC, the administration of IL-2 and IFN-α2a demon-strated high toxicity [72].

3.2. Th17 Cells and Proinflammatory Cytokines. The inflam-mation process is an important feature of any cancer [1]. Inregard to HPV-associated cancer, a deregulated proinflam-matory network is induced by HPV inducing a favourablemilieu for tumor development.

Th17 cell (CD4+, IL-17+), a T cell phenotype involved inthe inflammatory response, was reported to be linked tothe development of cervical cancer as well as others [73].It has been shown that the percentage of this cell pheno-type, as well as Th17/Treg ratio, was higher in peripheralblood samples of patients with premalignant and cervicalcancer lesions when compared to the normal cytologygroup [74], a similar outcome previously observed [75].In other studies, elevated levels of Th17 cells in CIN andcancer patients [73, 76] and in high-grade cervical lesions[77] were found, when compared to healthy controls. Ahigher statistical prevalence of this cell type was reportednot only in serum but also in the cervical tissue of cancerpatients. Likewise, a statistical significance of Th17 preva-lence among patients with advanced (higher prevalence)and early stages in malignant processes was observed[78] and thus, its level has been deemed a good indepen-dent prognostic factor in cervical cancer [79].

In HNSCC, elevated levels of Th17 cells were found alongwith Treg [80] in serum and in tumor milieu, concluding itsnegative impact on the immune response against HPV, espe-cially due to the induction of an exaggerated inflammatoryresponse through IL-17 secretion [81]. The increased levelsofTh17or IL-17were also found inpatientswith hypopharyn-geal carcinoma, as well as other cancers such as colon, gastricand lung, suggesting a connection with cancinogenesis andmalignant progression. Conversely, premalignant oral lesiontreatments with TGF-β type I receptor inhibitor and IL-23showedmaintenance of the Th17 phenotype instead of chang-ing toTreg cells. This resulted in the productionof stimulatoryand inflammatory molecules and slowed the progression ofpremalignant lesions to oral cancer [82]. The last research out-come demonstrated that the effect of Th17 is still unclear,mainly in other types of HPV-related cancers [79].

IL-1 (both IL-1α and -β) is the other interleukin presentin higher levels in cervical cancer. Secretion of IL-1 is pro-moted by keratinocyte damage, the major IL-1-producingcells [83]. This interleukin is also secreted by TAM, anotherimportant cell in harmful proinflammatory response whichinduces metastasis, tumor growth, angiogenesis, and Tregdifferentiation [21]. IL-1 expression is modulated by NK-κBand vice versa, and the same occurs with TNF-α, that partic-ipates in the IL-1 synthesis pathway [83].

In particular, IL-1β exhibits an essential role ininflammation-associated carcinogenesis and supports tumorgrowth and metastasis. This interleukin promotes the secre-tion of a great range of cytokines, chemokines, growth factors,and various metastatic mediators, such as TGF-β, VEGF,metalloproteinases, and endothelial adhesion molecules [84].In cancer studies, IL-1β is associated with a poor prognosis[84], and in cervical cancer research, it supports tumor pro-gression and carcinogenesis [85]. IL-1β was also overex-pressed in several types of tumors like breast, colon,oesophageal, lung, and oral cancer. A high throughput bioin-formatics analysis plus in vitro and in vivoobservationdemon-strated that IL-1β is one of the key genes involved in HNSCCformation. This interleukin is closely related to the malignanttransformation of oral cells, protumorigenic microenviron-ment generation that leads to oral carcinogenesis, and cellgrowth of the same type of cells [86]. Due to its crucial rolein inflammation and carcinogenesis, this interleukin has beenconsidered useful in therapeutic strategies [84] aswell as IL-1αwhich also plays an important role in carcinogenesis [87].Interestingly, the expression of IL-1α has been associated withhigher risk of distantmetastasis inHNSCC, themajor cause ofdeath in this type of cancer. In this scenario, the evaluation ofIL-1α and clinical information may predict patients with highrisk of HNSCC metastasis, thus leading to new treatmentstrategies [88].

Other examples of proinflammatory cytokines whichsupport inflammation-associated cervical carcinogenesis arelisted in Table 3.

3.3. Immunosuppressive Cytokines. There are several cyto-kines which are directly involved in downregulation ofinflammatory status, promoting infection progress andcancer development. These include TGF-β, IL-4, IL-6, andIL-10 which are the main Th2 cytokines related to this anti-inflammatory profile and are discussed in this topic.

The expression of these cytokines is modulated by HPVoncoproteins o create a Th2 microenvironment. They havebeen reported to be upregulated in premalignant and cancerlesions [41, 52], and were suggested as biomarkers for HPV-related cancer [96]. Other Th2 interleukins are also encoun-tered at high levels in cervical cancer patients, such as IL-9[97] and IL-15 [44]. The latter and TGF-β, for example, werereported to induce the expression of CD94/NKG2A, prevent-ing NK cell activity and CTL cytotoxicity [98]. An ongoingstudy evaluated a recombinant human IL-15 in advancedHNSCC patients in order to measure NK cell count, activity,and other immune response parameters (NCT01727076).

TGF-β plays a crucial role in the repression of immuneresponses against HPV and is upregulated in cervical



carcinogenesis by viral oncoprotein activities. It blocks effec-tor functions by suppressing antigen presentation, NK cyto-toxicity, B cell and CTL proliferation, and cytokinesynthesis of the Th1 profile. It downregulates IL-2 receptorsignalling in T cells and IL-12 expression by APCs. More-over, this cytokine is able to promote IL-10 expression bymacrophages, induces proteolytic activity, which causesangiogenesis and metastasis [43], induces CD94/NKG2Aexpression on T cells [98], and stimulates the differentiationof T cells to Treg and Th17 phenotypes [74]. Moreover,TGF-β upregulation has been associated with favouringtumor development, CIN III specimens, cervical cancer,and cancer invasiveness [43].

IL-4 is another important cytokine frequently mentionedin cervical cancer studies. It is able to inhibit cytotoxic activ-ity and IFN-γ expression, even in the presence of PMA(phorbol myristate acetate) and ionomycin—substances usedin carcinogenesis models that stimulate immune responses[99]. This interleukin induces a switch to a Th2 responsive-ness profile along with IL-2 [100] and is associated with viralpersistence, disease severity, and progression of precancerouslesions [43, 100].

Like IL-4, IL-6 is upregulated during cervical carcinogen-esis progression [90], playing a role in HPV-immortalizedand carcinoma-derived cervical cell line proliferation [101].It has been stated to induce the phosphorylation of STAT3(the activated condition) in HNSCC, causing immunosup-pression by inhibiting maturation of DC and activation ofneutrophil, macrophage, NK and T cells. STAT3 is a keytranscription factor which is involved in several otherimmunosuppressive activities such as IL-10 signalling,downregulation of IL-12, impairment of DC, and productionof Treg cells [102]. IL-6 also contributes to the proliferationand inhibition of apoptosis of cancer cells, and thus,supports chronic inflammation and cancer development[51]. This interleukin affects DC migration, induces

angiogenesis [103], MMP9 synthesis, and TAM differentia-tion [104]. However, IL-6 has also been reported to playanti-infection and antitumor functions [105]. Its expressionis associated with a poor clinical prognostic factor [73]and its transcription repression can be used in immuno-therapy approaches in cervical [106] and other HPV-related cancers [47].

IL-10 is the most studied Th2 cytokine with immuno-suppressive activity in HPV-related cancer. It is synthesizedby various cells, including Treg, Th2, M2 macrophages,APCs, and NK cells. This interleukin supports the creationof a microenvironment favourable to tumor developmentand it is the main cytokine having a Th2 role along withTGF-β. It hampers immune surveillance by blocking (i)antigen presentation by DC through the reduction ofMHC II, adhesion, and costimulatory molecules, (ii) thesynthesis of cytokines of the Th1 profile and (iii) the activ-ities of monocytes and NK cells. IL-10 also supportsimmunomodulation by inducing the differentiation of Tcells and macrophages to the Treg [43] and M2 profiles,respectively [107].

Furthermore, it has been demonstrated that IL-10 preju-dices antiviral immune responses, since it impairs Th1 profiledifferentiation, CD8+ cytotoxic response, and CD3+ expres-sion, which is essential in activating T cells. This interleukinalso causes the downregulation of MHC I and II on the sur-face of monocytes. Moreover, this molecule is able to upreg-ulate HPV16 E7 in cervical carcinoma cells in vitro, inducingtumor proliferation [43].

Despite the immunosuppressive activities cited above,there were disagreements over what high levels of IL-10 wererelated to, since it has been also associated with low gradelesions [66]. However, its immune regulatory activities havebeen well established. Elevated levels of IL-10 are commonlycorrelated with high-grade lesions and cancer condition, andits immunosuppressive roles have been reported countless

Table 3: Some proinflammatory cytokines in HPV-related carcinogenesis.

Cytokine Action mechanism

IL-8

(i) It induces neutrophil chemoattraction and cell survival [18](ii) It stimulates cell growth and cancer metastasis [89](iii) Prognosis of patients with high levels of IL-8 is extremely poor [48] and its

expression was associated with lesion severity [90]

IL-17

(i) It is associated with lymphatic metastasis [91](ii) It is found in high levels in patients with cervical cancer [91](iii) It is also linked to the antitumor response [92], when it supports the

recruitment and activation of neutrophils,the maturation of DC/priming of T cells, and the synthesis of TNF-α,IL-1β, and IL-6 [74, 75, 93]

IL-23

(i) It is synthesized by activated APCs [94](ii) It induces macrophage secretion of TNF-α, DC production of IL-12,

and the synthesis of IL-17 [95](iii) It induces upregulation of MMP9 (matrix metalloproteinase 9),

tumor angiogenesis, and TAM activity and prevents T CD8+ cell infiltration [95](iv) As well as IL-17, shows antitumor activities through its immune surveillance

properties, such as the promotion of CTL and NK cell, the IFN-γ secretion,and the stimulation of the IL-12-induced Th1 response [95]


times [42, 43, 52, 91], what are probably induced by HPV E2protein activity [43].

Hence, IL-10, along with CD8+ T cells and Treg cell ratio,has been considered an independent factor of poor progno-sis. Treg cell is associated with tumor growth, tumorigenesis,and lymph node metastasis, constituting a poor prognosis forpatients with cervical and other cancers as well [55]. Thesecells are rich in high-grade carcinoma samples, suppressCTL and NK cell activities, and support cancer progressionthrough cytokine synthesis, such as IL-10 itself and TGF-β[43, 80]. Therefore, Treg cells, IL-10, and TGF-β (and otherimmune activities interfered by HPV are summarized inFigure 5) are considered targets for therapeutic interventions.

Another cytotoxic cell has also been encountered at highnumbers in cervical cancer samples [109]. The expansion ofthis novel T cell subtype might affect lesion fate [110]. It ispositive for CD4 and NKG2D markers and is subdivided inCD28+/−, showing a statistically significant association withunderexpression of several proinflammatory cytokines, suchas IL1-β, IL-2, and TNF-α [109]. The role it plays andwhether it is related to tumor growth or tumor suppressionare still not known.

Novel cytokines in cervical cancer study have been gain-ing more and more attention due to their therapeutic poten-tial. One of them, IL-37, has shown promising results becauseof its anticancer activity. For the first time, its anticancer rolein an in vitro cervical cancer model has been demonstrated.This interleukin suppressed proliferation and invasion ofHeLa and C33A cell lines, with higher inhibition rates inHeLa cell line, showing that anticancer activities were relatedto the HPV. This occurred through the inhibition of mRNA

and protein expression of STAT3. Moreover, STAT3 phos-phorylation was also blocked. This protein is a key transducersignalling molecule for developing an immune response in atumor setting and could be an antagonist of IL-6 activitysince STAT3 is central to the IL-6 signalling pathway [111].

In summary, cytokines are key molecules which modulatethe pathologymilieu. Thus, the regulation of the transcription,the synthesis, and the secretion of these signalling molecules inimmunotherapies are necessary events to achieve satisfactoryresults in therapy. Cytokines are involved in several mecha-nisms of the immune system, such as immune cellmaturation/differentiation, maintaining activation and regu-lating immune cell activities, which contribute to an immuno-protective background. Consequently, these molecules areimportant for therapy, i.e., by supporting the establishmentof a proimmune milieu for infection clearance or by prevent-ing the immunosuppressive role of immune cells. Many ther-apeutic interventions which take advantage of this have beendeveloped and are currently in progress in therapeutic prac-tices with very promising results, such as the active cytokinecomponents of IRX-2, which caused an increase in antigenpresentation, in NK cell activity, in the synthesis of costimula-tory molecules and in CD8+ T cell responses [112]. The mix-ture of cytokines is safe, generally well tolerated, and hascurrently been tested in a phase II trial for HNSCC [38].

4. Future Prospects

The immune response is vital in HPV-related cancer diseaseprogression and resolution. In this set of collected data, thehost immune response was observed to be used to benefit

IL-12 Th2 Treg

Th1

IL-2

IFN-�훾

IFN-�훾

Lower CD8 activation

MHC-1 (HLA-A)TLR3, TLR9CCR7

TNF-�훼

TGF-�훽TGF-�훽IL-6IL-1�훽IL-17

pRbIL-10 CTLA-4

CD8 T cellTumor cell

hrHPV

CD4 T cell

PD-1E7 oncoprotein

Tumor cellproliferation

IL-1�훽p53apoptosis

IL-4IL-10

1 2 3

Figure 5: Interferences of HPV on CD4+/CD8+ lymphocyte responses, tumor proliferation, and apoptosis. (1) HPV is able to downregulatethe Th1 response by decreasing proinflammatory cytokines. Moreover, Th2 and Treg response are stimulated by the virus because anti-inflammatory cytokines are expressed in higher amounts in tumor microenvironment. (2) Tumor cells show different mechanisms fortheir activation and survival. HPV (especially E6 and E7 oncogenes) promotes changes in cell cycle regulatory genes (e.g., pRb and p53),leading to tumor cell proliferation. In addition, Th2 cytokines produced by APC and T CD4+ cells generate a chemical microenvironmentthat favours tumor establishment [2]. (3) T CD8+ lymphocyte activities are affected by HPV infection that induces a decreased antigenpresentation (MHC-I/HLA-A) and expression of TLRs and CCR7 on membrane surface of infected cells. Moreover, these cells also showupregulation of CTLA-4 and PD-1 inhibitor molecules [108].


patient’s health through various ways, as these attributesoccur in the natural resolution of infection. These newimmunological approaches open novel horizons in diagnosisand especially in cancer therapy. TLRs and cytokines havebeen used to create an ideal tumor milieu for preventingtumor development or favouring transformed cell destruc-tion. Their utilization appears to be a very promisingimmunotherapeutic strategy nowadays. The activation ofDC and NK cells by means of administration of appropri-ate TLR and cytokines is essential to ensure the T-helperand cytotoxic responses.

Another possibility of using TLRs and cytokines inimmunotherapy is their use in combination with monoclonalantibodies that prevent the activity of the immunecheckpoint molecules, such as CTLA-4, PD-1, LAG-3, andTIM-3. These receptors are found in T cells and interact toligands located at the cell membrane of tumor and antigen-presenting cells. In tumor pathogenesis, the activation ofthese molecules triggers signalling pathways which primarilyprevent the function of CD8+ and CD4+ cells. In addition,the induction of other immune evasion activities offers asuitable environment for infection persistence and tumordevelopment. Thus, inhibition of these activities may beconsidered a good therapeutic target; CTLA-4, for example,is found to be highly expressed in HPV-related cancerwhen compared to HPV-negative samples [22]. It competeswith CD28 for interactions with the CD80 and CD86ligands in DC surface, which prevented T cell priming bythese cells. PD-1/PD-L1 is also greatly related to immuneescape in HPV-related cancers, being correlated with areduced disease survival rate due to CTL activity attenua-tion [102, 113]. Similarly, LAG-3 and TIM-3 have beenrecently considered in immunotherapy approaches. Thefirst molecule enhances Treg cell activity [102]; and the sec-ond one induces T cell exhaustion and suppression of theinnate response, associated with poor prognosis and tumorprogression [114].

Checkpoint inhibitor monoclonal antibodies combinedwith other immune approaches have already been evaluatedfor cervical and HNSCC treatment: anti-PD-1 such as pem-brolizumab (which is in ongoing studies for HNSCC treat-ment: NCT02255097 and NCT02252042) and nivolumab(NCT02105636 and NCT02488759); anti-PD-L1 such asdurvalumab (NCT02207530) [46]; anti-CTLA4 such as ipili-mumab and tremelimumab; anti-LAG-3 such as BMS-986016 (NCT01968109); and anti-TIM-3 (NCT02817633).The cited studies including LAG-3 and TIM-3 were for treat-ment of solid tumors. On the other hand, the combinationwith TLR agonists is new for HPV-related cancers; thereare only two studies, NCT02643303 and NCT02124850,which are currently recruitingpatients for testing thepoly(I:C)with tremelimumab and durvalumab and the combination ofVTX-2337 plus cetuximab or VTX-2337 plus cetuximab plusnivolumab, respectively. Regarding the evaluation of thecombination of checkpoint inhibitors with cytokines inHPV-related cancers, no current study is reported at least tothebest ofourknowledge; andonly three studieswere reportedfor other HPV-unrelated solid tumors (NCT02614456,NCT02174172, and NCT02947165).

Therefore, the combination of different immunothera-peutic methods has shown increased beneficial effects andseems to be crucial in achieving better outcomes as observedin preclinical and clinical trials. As discussed here, HPV-related tumors require a great immune suppressor statusfor cancer development with increased activities of Treg,CTLA-4, and PD-1 and the suppression of APC and NK cells.Thus, studies on such evasion mechanisms are needed andoffer new therapeutic perspectives.

Abbreviations

APC: Antigen-presenting cellCCL2/MCP-1: C-C motif chemokine ligand

2/monocyte chemoattractant protein-1CCL20/MIP-3α: C-C motif chemokine ligand

20/macrophage inflammatory protein-3αCCR7: C-C motif chemokine receptor 7CTL: cytotoxic T lymphocyte (CD8 T

lymphocyte)CTLA-4: Cytotoxic T lymphocyte-associated

antigen 4DAMP: Damage- or danger-associated molecular

patternsDC: Dendritic cellEGFR: Epidermal growth factor receptorHDAC1: Histone deacetylase 1HeLa: Human cervical cancer cell lineHNSCC: Head and neck squamous cell carcinomaHPV: human papillomavirusIκBα: Nuclear factor of kappa light polypeptide

gene enhancer inB-cell inhibitor, alpha

IRAK: IL-1R-associated kinaseIRF: Interferon regulatory factorISGF3: Interferon-stimulated gene factor 3ISRE: Interferon response elementIKK: Inhibitor of kappa B kinaseJARID1B: Lysine-specific demethylase 5BLC: Langerhans cellLPS: LipopolysaccharidesMHC: Major histocompatibility complexMMP9: Matrix metalloproteinase 9MPL: monophosphoryl lipid AMyD88: Myeloid differentiation primary response

protein 88NF-κB: Nuclear transcription factor

kappa BNK: Natural killerOPSCC: Oropharyngeal squamous cell carcinomaOSCC: oral squamous cell carcinomaPAMPs: Pathogen-associated molecular patternsPD-1: Programmed death-1 receptorPD-L1: Programmed death ligand-1PMA: phorbol myristate acetatePoly(I:C): Polyriboinosinic-polyribocytidylic acidPolyICLC: Modified version of poly(I:C) stabilized by

lysine and carboxymethylcelluloseRIP-1: Receptor interacting protein kinase-1














SCC: squamous cell carcinomaTAB: TGF-β-activated kinase

I/MAP3K7-binding proteinTAK: TNF receptor-associated factorTAM: Tumor-associated macrophage (M2

macrophage phenotype)TAP-1: Transporter antigen processing-1TIRAP: TIR-associated proteinTNF-α: Tumor necrosis factor αTLR: Toll-like receptorTRAM: Toll receptor-associated moleculeTRAF: TNF receptor-associated factorTRIF: TIR-domain-containing

adapter-inducing interferon-β.

Conflicts of Interest

The authors declare that there is no conflict of interestregarding the publication of this paper.

Authors’ Contributions

Aldo Venuti and Antonio Carlos de Freitas equallycontributed to this work.

Acknowledgments

The authors would like to thank Tania Merlino for Englishediting and corrections. This paper has received fundingfrom CNPq/edital PVE (401305/2014-7) and FACEPE/editalPRONEM (APQ-0562-2.02/2014).

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